In the previous annual report we studied the effects of aggregates and fragments on the potency of Hepatitis B Immune Globulin (HBIG). Three commercial HBIG lots and an FDA standard (ref.#2) were assayed by two methods, i.e., AUSAB method [to measure the bivalent intact IgG and the fragment (Fab')2] and a radioimmuno-precipitation (RIP) method (to measure the bivalent antibodies as well as the monovalent Fab/Fc and Fab fragments). Our results indicate that the polymer fractions are less potent, monomer and dimer fractions are equally active, and Fab/Fc enriched fractions may be inhibitory to AUSAB. We have since isolated and purified monomer and its fragments from one of the highly fragmented HBIG lots by both gel filtration and protein G chromatography. Fab/Fc, however, was only partially purified since we were unable to separate it completely from IgG monomer. When assayed with AUSAB, either Fab/Fc or Fab inhibited the potency of the ref.#2 monomer while Fc did not inhibit even when present at a high concentration. Furthermore, the monomer isolated from the highly fragmented lot was less potent than the monomer from HBIG ref.#2 which contained aggregates but no fragments. When subjected to IgG subclass analysis, the former monomer was found to contain largely IgG2 and very little IgG1 while the ref.#2 monomer contained largely IgG1. Subclass fractionation for ref.#2 was carried out by Protein A Sepharose chromatography with pH gradient elution. Although the method can not separate IgG4 efficiently from either IgG2 or IgG1, the IgGl enriched fraction possessed the highest anti-HBs specific activity while IgG2 or IgG3 fractions had low activities. Hence, fragmentation, inhibition by fragments, and loss of IgG1 are factors affecting the potency of HBIG. Work is still in progress.